All antibodies (Abs) are glycoproteins and all heavy chains have at least one N-linked carbohydrate in their constant regions. Many Abs also have variable (V) region associated carbohydrate. The proposed studies will systematically investigate the role and function of the Ab associated carbohydrate by altering its presence, position and structure. Central to this investigation is the ability to produce large quantities of antibodies possessing altered carbohydrates of defined structure using the well- characterized glycosylation mutants of Chinese hamster ovary (CHO) cells. A significant percentage of Abs contain variable region carbohydrate and although V region carbohydrate can influence Ag binding, the function of V region carbohydrate remains incompletely understood. A family of Abs of the same specificity with carbohydrate at different positions in their V region will be produced and their functional properties including in vitro half-life, reactivity with rheumatoid factors (RFs) and affinity for antigen (Ag) determined. Site-directed mutations will be used to ascertain which V region sequences can be glycosylated. The presence of carbohydrate in Ch2 is essential for the biological activity of IgG. The carbohydrate is always biantennary with heterogeneity in structure normally observed. Evidence suggests that certain glycosylation patterns can contribute to aberrant immunoglobulin activity with a high incidence of agalactosylated Abs observed in rheumatoid arthritis (RA). Abs with altered carbohydrate structure will be produced to determine if changes in carbohydrate structure result in changes in Ab function including half-life, the ability to bind cellular receptors for Fc and to activate complement by either the classical pathway or through mannose binding proteins. Recognition by lectins, monoclonal antibodies, and Proteins A and G, will be used to probe alterations in peptide conformation and carbohydrate accessibility. In addition to giving insights into Ab function, these experiments will elucidate the role of glycosylation alterations in creating pathogenic Abs. IgA is the important isotype in providing specific protection at the mucosal surface. The role of carbohydrate in IgA function will be investigated by altering both the presence and structure of its associated N-linked carbohydrate. The contribution of the O-linked carbohydrate of IgA1 will also be assessed and the role of the N-linked carbohydrate of J- chain investigated. The recombinant molecules will be evaluated for their in vivo half-life and biodistribution as well as their resistance to bacterial proteases, binding to bacterial and cellular receptors, ability to activate the alternative complement pathway and to bind to the polymeric Ig receptor and be transported across the epithelium. The information obtained may permit the design and production of IgA more effective in passive protection.